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Spectral library generation mlazear  2024-09-23 12:39
 

Hi,

Is there a way to construct a spectral library such that Skyline will just "trust" the user that parameters are correct? I am trying to build some empirical (e.g. constructed from DDA) spectral libraries for DIA runs where the peptides have exotic/unlocalized modifications - and I am really struggling with how to get Skyline to accept a spectral library.

Ideally, I can just pass in a list of arbitrary ID, precursor m/z, precursor charge, product m/z, library intensity and have Skyline accept it without any "error" checking (e.g. do not try to match the product m/z to the peptide sequence). Is there a way I can fool Skyline into accepting such a list? I don't want to have to manually add the modifications to Skyline, since I have 1000s of them. I am happy to manually construct a library in whatever input format is needed.
 
 
Nick Shulman responded:  2024-09-23 13:01
I could imagine a way of doing this by creating a .ssl file and a .ms2 file.
You can learn more about .ssl ("spectrum sequence list") and .ms2 files here:
https://skyline.ms/wiki/home/software/BiblioSpec/page.view?name=BiblioSpec%20input%20and%20output%20file%20formats

It sounds like you will run into a problem when you try to use this library in Skyline if you do not want to tell Skyline about all of your modifications.
The modifications in a .blib spectral library are always described only by their masses, but Skyline always requires you to provide more information about the modification (at least to give the modification a unique name) in order to be able to insert a peptide with that modification into your Skyline document.

If you send us your files, we might be able to figure out a solution for you. If you send me your list of thousands of modifications I could probably give you a Skyline document which contains all of your modifications. If you send us your list of precursor and product m/z's etc I might be able to show you how to create .ssl and .ms2 files.

If you want to have peptides in your document which have modifications that Skyline does not understand, then you might want to just treat them as small molecules.

Files which are less than 50MB can be attached to these support requests. You can always upload larger files here:
https://skyline.ms/files.url
-- Nick
 
mlazear responded:  2024-09-23 13:23
If they can be programmatically added, then no problem with listing out the modifications - I just don't want to have to click in a GUI that much :). I think the bigger issue I've run into is getting Skyline to understand un-localized modifications (e.g. may be a superposition/mixture of different positions) - I've attached an example precursor.